High threonine derivatives of α-hordothionin

ABSTRACT

Derivatives of α-hordothionin made by position-specific substitution with threonine residues provide threonine in plants.

This is a continuation of application Ser. No. 08/459,180, filed Jun. 2,1995 now abandoned.

TECHNICAL FIELD

This invention relates to the improvement of feed formulations.Specifically, this invention relates to derivatives of α-hordothioninwhich provide higher percentages of threonine in plants.

BACKGROUND OF THE INVENTION

Feed formulations are required to provide animals essential nutrientscritical to growth. However, crop plants are generally rendered foodsources of poor nutritional quality because they contain low proportionsof several amino acids which are essential for, but cannot besynthesized by, animals.

For many years researchers have attempted to improve the balance ofessential amino acids in the proteins of important crops throughbreeding programs. As more becomes known about storage proteins and theexpression of the genes which encode these proteins, and astransformation systems are developed for a greater variety of plants,molecular approaches for improving the nutritional quality of seedproteins can provide alternatives to the more conventional approaches.Thus, specific amino acid levels can be enhanced in a given crop viabiotechnology.

One alternative method is to express a heterologous protein of favorableamino acid composition at levels sufficient to obviate food or feedsupplementation. For example, a number of seed proteins rich in sulfuramino acids have been identified. A key to good expression of suchproteins involves efficient expression cassettes with seed specificpromoters. Not only must the gene-controlling regions direct thesynthesis of high levels of mRNA, the mRNA must be translated intostable protein.

Among the essential amino acids needed for animal nutrition, oftenmissing from crop plants, are methionine, threonine and lysine. Attemptsto increase the levels of these free amino acids by breeding, mutantselection and/or changing the composition of the storage proteinsaccumulated in crop plants has met with minimal success. Usually, theexpression of the transgenic storage protein was too low. Thephaseolin-promoted Brazil nut 2S expression cassette is an example of aneffective chimeric seed-specific gene. However, even though Brazil nutprotein increases the amount of total methionine and bound methionine,thereby improving nutritional value, there appears to be a thresholdlimitation as to the total amount of methionine that is accumulated inthe seeds. The seeds remain insufficient as sources of methionine.

An alternative to the enhancement of specific amino acid levels byaltering the levels of proteins containing the desired amino acid ismodification of amino acid biosynthesis. Recombinant DNA and genetransfer technologies have been applied to alter enzyme activitycatalyzing key steps in the amino acid biosynthetic pathway. Glassman,U.S. Pat. No. 5,258,300; Galili, et al., European Patent Application No.485970; (1992); incorporated herein in its entirety by reference.However, modification of the amino acid levels in seeds is not alwayscorrelated with changes in the level of proteins that incorporate thoseamino acids. Burrow, et al., Mol. Gen. Genet.; Vol. 241; pp. 431-439;(1993); incorporated herein in its entirety by reference. Althoughsignificant increases in free lysine levels in leaves have been obtainedby selection for DHDPS mutants or by expressing the E. coli DHDPS inplants, it remains to be shown that these alterations can increase boundtarget amino acids, which represent some 90% or more of total aminoacids. Thus, there is minimal impact on the nutritional value of seeds.

Based on the foregoing, there exists a need for methods of increasingthe levels of the essential amino acids, threonine, methionine andlysine in seeds of plants.

It is therefore an object of the present invention to provide methodsfor genetically modifying plants to increase the levels of the essentialamino acid threonine in the plants.

It is a further object of the present invention to provide seeds forfood and/or feed with higher levels of the essential amino acidthreonine than wild species of the same seeds.

DISCLOSURE OF THE INVENTION

It has now been determined that one class of compounds, theα-hordothionins, can be modified to enhance their content of threonine.α-hordothionin is a 45-amino acid protein which has been wellcharacterized. It can be isolated from seeds of barley (Hordeumvulgare). The molecule is stabilized by four disulfide bonds resultingfrom eight cysteine residues. The amino acid sequence is as provided inSEQUENCE I.D. No.1. In its native form, it is especially rich inarginine and lysine residues, containing 5 residues (10%) of each.However, it contains only 3 residues (7%) of the essential amino acidthreonine.

The protein has been synthesized and the three-dimensional structuredetermined by computer modeling. The modeling of the protein predictsthat the ten charged residues (arginine at positions 5,10,17,19 and 30,and lysine at positions 1,23,32,38 and 45) all occur on the surface ofthe molecule. The side chains of the polar amino acids (asparagine atposition 11, glutamine at position 22 and threonine at position 41) alsooccur on the surface of the molecule. Furthermore, the hydrophobic aminoacids (such as the side chains of leucine at positions 8,15 24 and 33and valine at position 18) are also solvent-accessible.

Three-dimensional modeling of the protein indicates that the arginineresidue at position 10 is critical to retention of the appropriate3-dimensional structure and possible folding through hydrogen bondinteractions with the C-terminal residue of the protein. A threoninesubstitution at that point would disrupt the hydrogen bonding involvingarginine at position 10, serine at position 2 and lysine at position 45,leading to destabilization of the structure. The synthetic peptidehaving this substitution could not be made to fold correctly, whichsupported this analysis. Conservation of the arginine residue atposition 10 provided a protein which folded correctly.

Since threonine is a polar amino acid, the surface polar amino acidresidues, asparagine at position 11 and glutamine at position 22, weresubstituted; and the charged amino acids, lysine at positions 1,23,32and 38 and arginine at positions 5,17,19, and 30, were substituted withthreonine. The resulting compound has the sequence indicated in SEQUENCEI.D. No. 2. The molecule can be synthesized by solid phase peptidesynthesis and folds into a stable structure. It has 13 threonineresidues (29%).

While SEQUENCE I.D. No. 2 is illustrative of the present invention, itis not intended to be a limitation. Threonine substitutions can also beperformed at positions containing charged amino acids. Only arginine atposition 10 and lysine at position 45 are critical for maintaining thestructure of the protein. One can also substitute at the sites havinghydrophobic amino acids. These include positions 8,15,18 and 24. Theresulting compound has the sequence indicated in SEQUENCE I.D. NO. 3.

Synthesis of the compounds is performed according to methods of peptidesynthesis which are well known in the art and thus constitute no part ofthis invention. In vitro, the compounds have been synthesized on anapplied biosystems model 431a peptide synthesizer using fastmoc™chemistry involving hbtu(2-(lh-benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate, as published by Rao, et al., Int. J. Pep. Prot.Res.; Vol. 40; pp. 508-515; (1992); incorporated herein in its entiretyby reference. Peptides were cleaved following standard protocols andpurified by reverse phase chromatography using standard methods. Theamino acid sequence of each peptide was confirmed by automated edmandegradation on an applied biosystems 477a protein sequencer/120a pthanalyzer. More preferably, however, the compounds of this invention aresynthesized in vivo by bacterial or plant cells which have beentransformed by insertion of an expression cassette containing asynthetic gene which when transcribed and translated yields the desiredcompound. Such empty expression cassettes, providing appropriateregulatory sequences for plant or bacterial expression of the desiredsequence, are also well-known, and the nucleotide sequence for thesynthetic gene, either RNA or DNA, can readily be derived from the aminoacid sequence for the protein using standard reference texts.Preferably, such synthetic genes will employ plant-preferred codons toenhance expression of the desired protein.

Industrial Applicability

The following description further exemplifies the compositions of thisinvention and the methods of making and using them. However, it will beunderstood that other methods, known by those of ordinary skill in theart to be equivalent, can also be employed.

Plants

The genes which code for these compounds can be inserted into anappropriate expression cassette and introduced into cells of a plantspecies. Thus, an especially preferred embodiment of this methodinvolves inserting into the genome of the plant a DNA sequence codingfor a compound of this invention in proper reading frame, together withtranscription initiator and promoter sequences active in the plant.Transcription and translation of the DNA sequence under control of theregulatory sequences causes expression of the protein sequence at levelswhich provide an elevated amount of the protein in the tissues of theplant.

Preferred plants that are to be transformed according to the methods ofthis invention are cereal crops, including maize, rye, barley, wheat,sorghum, oats, millet, rice, triticale, sunflower, alfalfa, rapeseed andsoybean.

Synthetic DNA sequences can then be prepared which code for theappropriate sequence of amino acids, and this synthetic DNA sequence canbe inserted into an appropriate plant expression cassette.

Likewise, numerous plant expression cassettes and vectors are well knownin the art. By the term "expression cassette" is meant a complete set ofcontrol sequences including initiation, promoter and terminationsequences which function in a plant cell when they flank a structuralgene in the proper reading frame. Expression cassettes frequently andpreferably contain an assortment of restriction sites suitable forcleavage and insertion of any desired structural gene. It is importantthat the cloned gene have a start codon in the correct reading frame forthe structural sequence.

In addition, the plant expression cassette preferably includes a strongconstitutive promoter sequence at one end to cause the gene to betranscribed at a high frequency, and a poly-a recognition sequence atthe other end for proper processing and transport of the messenger RNA.An example of such a preferred (empty) expression cassette into whichthe cDNA of the present invention can be inserted is the pPHI414 plasmiddeveloped by Beach, et al., of Pioneer Hi-Bred International, Inc.,Johnston, Iowa, as disclosed in U.S. patent application Ser. No.07/785,648, (1991); incorporated herein in its entirety by reference.Highly preferred plant expression cassettes will be designed to includeone or more selectable marker genes, such as kanamycin resistance orherbicide tolerance genes.

By the term "vector" herein is meant a DNA sequence which is able toreplicate and express a foreign gene in a host cell. Typically, thevector has one or more endonuclease recognition sites which may be cutin a predictable fashion by use of the appropriate enzyme such vectorsare preferably constructed to include additional structural genesequences imparting antibiotic or herbicide resistance, which then serveas markers to identify and separate transformed cells. Preferredmarkers/selection agents include kanamycin, chlorosulfuron,phosphonothricin, hygromycin and methotrexate. A cell in which theforeign genetic material in a vector is functionally expressed has been"transformed" by the vector and is referred to as a "transformant."

A particularly preferred vector is a plasmid, by which is meant acircular double-stranded DNA molecule which is not a part of thechromosomes of the cell.

As mentioned above, both genomic and cDNA encoding the gene of interestmay be used in this invention. The vector of interest may also beconstructed partially from a cDNA clone and partially from a genomicclone. When the gene of interest has been isolated, genetic constructsare made which contain the necessary regulatory sequences to provide forefficient expression of the gene in the host cell. According to thisinvention, the genetic construct will contain (a) a first geneticsequence coding for the protein or trait of interest and (b) one or moreregulatory sequences operably linked on either side of the structuralgene of interest. Typically, the regulatory sequences will be selectedfrom the group comprising of promoters and terminators. The regulatorysequences may be from autologous or heterologous sources.

Promoters that may be used in the genetic sequence include NOS, OCS andCaMV promoters.

An efficient plant promoter that may be used is an overproducing plantpromoter. Overproducing plant promoters that may be used in thisinvention include the promoter of the cholorophyll α-βbinding proteinand the promoter of the small sub-unit (ss) of theribulose-1,5-biphosphate carboxylase from soybean. See e.g. Berry-Lowe,et al., J. Molecular and App. Gen.; Vol. 1; pp. 483-498; (1982);incorporated herein by reference. These two promoters are known to belight-induced, in eukaryotic plant cells. See e.g., An AgriculturalPerspective, A. Cashmore, Pelham, New York; pp. 29-38; (1983); G.Coruzzi, et al., J. Biol. Chem.; Vol. 258; p. 1399; (1983); and P.Dunsmuir, et al., J. Molecular and App. Gen.; Vol. 2; p. 285; (1983);all incorporated herein by reference.

The expression cassette comprising the structural gene for the proteinof this invention operably linked to the desired control sequences canbe ligated into a suitable cloning vector. In general, plasmid or viral(bacteriophage) vectors containing replication and control sequencesderived from species compatible with the host cell are used. The cloningvector will typically carry a replication origin, as well as specificgenes that are capable of providing phenotypic selection markers intransformed host cells. Typically, genes conferring resistance toantibiotics or selected herbicides are used. After the genetic materialis introduced into the target cells, successfully transformed cellsand/or colonies of cells can be isolated by selection on the basis ofthese markers.

Typically, an intermediate host cell will be used in the practice ofthis invention to increase the copy number of the cloning vector. Withan increased copy number, the vector containing the gene of interest canbe isolated in significant quantities for introduction into the desiredplant cells. Host cells that can be used in the practice of thisinvention include prokaryotes, including bacterial hosts such as E.coli, S. typhimurium, and Serratia marcescens. Eukaryotic hosts such asyeast or filamentous fungi may also be used in this invention. Sincethese hosts are also microorganisms, it will be essential to ensure thatplant promoters which do not cause expression of the protein in bacteriaare used in the vector.

The isolated cloning vector will then be introduced into the plant cellusing any convenient technique, including electroporation (inprotoplasts), retroviruses, bombardment, and microinjection into cellsfrom monocotyledonous or dicotyledonous plants in cell or tissue cultureto provide transformed plant cells containing as foreign DNA at leastone copy of the DNA sequence of the plant expression cassette.Preferably, the monocotyledonous species will be selected from maize,sorghum, wheat or rice, and the dicotyledonous species will be selectedfrom soybean, alfalfa, rapeseed, sunflower or tomato. Using knowntechniques, protoplasts can be regenerated and cell or tissue culturecan be regenerated to form whole fertile plants which carry and expressthe gene for a protein according to this invention. Accordingly, ahighly preferred embodiment of the present invention is a transformedmaize plant, the cells of which contain as foreign DNA at least one copyof the DNA sequence of an expression cassette of this invention.

It will also be appreciated by those of ordinary skill that the plantvectors provided herein can be incorporated into agrobacteriumtumefaciens, which can then be used to transfer the vector intosusceptible plant cells, primarily from dicotyledonous species. Thus,this invention provides a method for increasing threonine levels inagrobacterium tumefaciens-susceptible dicotyledonous plants in which theexpression cassette is introduced into the cells by infecting the cellswith agrobacterium tumefaciens, a plasmid of which has been modified toinclude a plant expression cassette of this invention.

    __________________________________________________________________________    SEQUENCE LISTING    (1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 3    (2) INFORMATION FOR SEQ ID NO:1:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 45 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    LysSerCysCysArgSerThrLeuGlyArgAsnCysTyrAsnLeuCys    151015    ArgValArgGlyAlaGlnLysLeuCysAlaGlyValCysArgCysLys    202530    LeuThrSerSerGlyLysCysProThrGlyPheProLys    354045    (2) INFORMATION FOR SEQ ID NO:2:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 45 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    ThrSerCysCysThrSerThrLeuGlyArgThrCysTyrAsnLeuCys    151015    ThrValThrGlyAlaThrThrLeuCysAlaGlyValCysThrCysThr    202530    LeuThrSerSerGlyThrCysProThrGlyPheProLys    354045    (2) INFORMATION FOR SEQ ID NO:3:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 45 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    ThrSerCysCysThrSerThrThrGlyLysThrCysTyrAsnThrCys    151015    ThrThrThrArgAlaThrThrThrCysAlaGlyValCysThrCysThr    202530    LeuThrSerSerGlyThrCysProThrGlyPheProLys    354045    __________________________________________________________________________

What is claimed is:
 1. A protein having the sequence of SEQUENCE I.D.No. 3 wherein the amino acid residues at one or more of positions1,5,7,8,11,15,17,18,19,22,23,24,30,32,34,38 and 41 are threonine, andthe remainder of the residues at those positions are the residues at thecorresponding positions in SEQUENCE I.D. No.
 1. 2. The protein of claim1 wherein one or more of the amino acid residues at1,5,7,11,17,19,22,23,30,32,34,38 and 41 are threonine.
 3. The protein ofclaim 2 wherein at least 5 of the amino acid residues at positions1,5,7,11,17,19,22,23,30,32,34,38 and 41 are threonine.
 4. The protein ofclaim 3 wherein at least 7 of the amino acid residues at positions1,5,7,11,17,19,22,23,30,32,34,38 and 41 are threonine.
 5. A nucleotidesequence which codes for a protein having the sequence of SEQUENCE I.D.No. 3 wherein the amino acid residues at one or more of positions1,5,7,8,11,15,17,18,19,22,23,24,30, 32,34,38 and 41 are threonine, andthe remainder of the residues at those positions are the residues at thecorresponding positions in SEQUENCE I.D. No.
 1. 6. An RNA sequence whichcodes for a protein having the sequence of SEQUENCE I.D. No. 3 whereinthe amino acid residues at one or more of positions1,5,7,8,11,15,17,18,19,22,23,24,30, 32,34,38 and 41 are threonine, andthe remainder of the residues at those positions are the residues at thecorresponding positions in SEQUENCE I.D. No.
 1. 7. A DNA sequence whichcodes for a protein having the sequence of SEQUENCE I.D. No. 3 whereinthe amino acid residues at one or more of positions1,5,7,8,11,15,17,18,19,22,23,24,30,32,34,38 and 41 are threonine, andthe remainder of the residues at those positions are the residues at thecorresponding positions in SEQUENCE I.D. No.
 1. 8. An expressioncassette containing the DNA sequence of claim 7 operably linked to plantregulatory sequences which cause the expression of the DNA sequence inplant cells.
 9. A bacterial transformation vector comprising anexpression cassette according to claim 8, operably linked to bacterialexpression regulatory sequences which cause replication of theexpression cassette in bacterial cells.
 10. Bacterial cells containingas a foreign plasmid at least one copy of a bacterial transformationvector according to claim
 9. 11. Transformed plant cells containing atleast one copy of the expression cassette according to claim
 8. 12. Thetransformed plant cells of claim 11, wherein the cells are of amonocotyledonous species.
 13. The transformed plant cells of claim 12,wherein the cells are selected from the group consisting of maize,sorghum, wheat and rice cells.
 14. The transformed cells of claim 11,wherein the cells are of a dicotyledonous species.
 15. The transformedcells of claim 14, wherein cells are selected from the group consistingof soybean, alfalfa, rapeseed, sunflower, tobacco and tomato cells. 16.A maize cell or tissue culture comprising cells according to claim 13.